The cover of the heart, or epicardium, consists of a single layer of mesothelial cells. During cardiac development, epicardial cells undergo Epithelial-to-Mesenchymal Transition (EMT) to form multipotent precursors known as epicardial-derived cells (EPDC). The EPDC migrate into myocardial tissue (containing cardiomyocytes) and subsequently differentiate into fibroblasts, myofibroblasts, and smooth muscle cells. In adult hearts, a similar process of epicardial cell proliferation, migration, and differentiation occurs after myocardial infarction (MI, heart attack). EPDC differentiation into vascular endothelial cells or cardiomyocytes is rare and not well understood. Recently, we observed that running (exercise) in mice promotes differentiation of EPDC into microvascular endothelial cells (CD31+). After running, EPDC appear to generate endothelial cells and not other cardiac cell types. Of interest, running promotes cardiac hypertrophy that requires additional perfusion (blood flow) and may therefore stimulate the contribution of EPDC to capillaries. We hypothesized that running exercise induces gene expression in epicardial cells that promotes endothelial specification. To test our hypothesis, we developed an efficient method to directly isolate primary adult epicardial cells from the heart cover based on their expression of integrin-β4 (CD104). After 2 hours of protease digestion, we used Magnetic-Activated Cell Sorting with antibodies against CD104 (CD104 MACS) to obtain undifferentiated epicardial cells; this was confirmed by expression of Keratin-18, an epicardial-specific protein in the heart. By cDNA microarray assays and bioinformatics analysis, we compared the gene expression profile of epicardial cells isolated from running-conditioned mice with that of age-matched controls (non-runners). Our data suggest that extracellular matrix remodeling in the heart is mediated, in part, by epicardial cells during running. Furthermore, we identify epicardial gene expression for cell signals/pathways and transcription factors that may enhance vascular perfusion after MI through promoting angiogenesis or endothelial specification of epicardial derivatives.

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Age-related macular degeneration (AMD) is a leading cause of irreversible blindness in the Western world. Although, the majority of stem cell research to date has focused on production of RPE and photoreceptor cells for the purpose of evaluating disease pathophysiology and cell replacement, there is strong evidence that the choroidal endothelial cells (CECs) that form the choriocapillaris vessels are the first to be affected in this disease. As such, to accurately evaluate disease pathophysiology and develop an effective treatment, production of patient-specific stem cell-derived CECs will be required. During the first stage of my Ph.D work, represented in Chapter 1 of this dissertation, I developed a co-culture system to differentiate mouse stem cells into CECs. I reprogrammed dermal fibroblasts from the Tie2-GFP mouse into two independent iPSC lines. TheTie2-GFP iPSCs were differentiated into CECs using a co-culture method with either the monkey RF/6A CEC line or primary mouse CECs. IPSC-derived CECs were characterized via rt-PCR and immunocytochemistry (ICC) for EC- and CEC-specific markers. The mouse iPSC-derived CECs described in Chapter 1 expressed the CEC-specific marker carbonic anhydrase IV (CA4), eNOS, FOXA2, PLVAP, CD31, CD34, ICAM-1, Tie2, TTR, VE-cadherin, and vWF. These Tie2-GFP iPSC-derived CECs paved the way for the rest of my Ph.D, in which I transitioned into using human iPSCs to generate patient-specific CECs. During the second phase of my graduate work, presented in Chapter 3, I developed a novel stepwise differentiation protocol suitable for generating human iPSC-derived CECs. I used previously published RNA-seq data of the monkey CEC line, RF/6A and two statistical screens to develop media comprised of various protein combinations. In both screens, I identified connective tissue growth factor (CTGF) as the key component required for driving CEC development. I also found that a second factor, called TWEAKR, promoted iPSC to CEC differentiation by inducing endogenous CTGF secretion. CTGF-driven iPSC-derived CECs formed capillary tube-like vascular networks, and expressed the EC-specific markers CD31, ICAM1, PLVAP, vWF, and the CEC-restricted marker CA4. These patient-specific iPSC-derived CECs made it possible for me to proceed into the next phase of my Ph.D work, in which I started working with AMD patient-specific iPSC-derived CECs to evaluate AMD pathophysiology. In the final stage of my Ph.D, represented in Chapter 4, I used the novel CEC differentiation method I developed to generate AMD iPSC-derived CECs and use these cells for AMD disease modeling. In line with previous studies that the membrane attack complex (MAC) forms in the AMD choriocapillaris, I showed that the AMD iPSC-derived CECs were much more susceptible to MAC formation and cell death when the cells were antagonized with complement components. I also demonstrated that, unlike the control CECs, the AMD CECs lost their capillary tube-like structures when the cells were cultured for over ten days, indicating that the AMD CECs may also exhibit other disease phenotypes other than susceptibility to MAC and cytolysis. Overall, the work I present in this dissertation will help push the AMD research field forward by providing a way to directly study AMD patient-specific iPSC-derived CECs and how they differ from healthy iPSC-derived CECs. In combination with RPE and photoreceptor cells, these patient-specific iPSC-CECs will make it possible to study AMD patient-specific CECs in vitro to better understand AMD pathogenesis and to develop autologous cell replacement therapies to replenish patients’ damaged choroids with healthy CECs.

Age-related macular degeneration (AMD) is a devastating disease causing vision loss in millions of people around the world. Loss of choroidal endothelial cells (CECs) is one of the earliest detectable events in AMD, and, because the outer retina relies on the choriocapillaris for metabolic support, this loss may be the trigger for progression to more advanced stages. A crucial event that occurs in the aging choriocapillaris is accumulation of the membrane attack complex (MAC), which may result in complement-mediated CEC lysis, and may be a primary cause for AMD-associated choriocapillaris degeneration. Previous studies have also shown the accumulation of C-reactive protein (CRP) in the choriocapillaris in eyes with AMD and those with the high-risk CFH genotype. While both CRP and the MAC have been implicated in AMD, the precise contribution of these molecules to disease pathophysiology has not been fully elucidated. Furthermore, there is a critical need to better understand the causes for pathologic changes to CECs during AMD and to establish methods for treatment in cases where CECs have already been lost. Therefore, the goals of this thesis are 1) to investigate the role of CRP and complement activation in AMD pathogenesis, and 2) to develop an in vitro method to study CEC replacement strategies. To address these questions, we first evaluated MAC levels in the choriocapillaris in comparison to 19 other tissues throughout the human body in order to determine in which tissues MAC accumulates with normal aging. Interestingly, we found that the choriocapillaris was the only tissue with high levels of the MAC, which was not detected in any of the other tissues. The restricted accumulation of MAC in the choriocapillaris may, in part, explain the specificity of AMD to the neural retina, RPE and choroid, and the relative absence of systemic pathology in this disease. We then studied genotyped human donor eyes and found that eyes homozygous for the high-risk CFH (Y402H) allele had elevated monomeric CRP (mCRP) within the choriocapillaris and Bruch's membrane, compared to those with the low-risk genotype. In order to assess the physiological effects mCRP has on CECs in vitro, CECs and organ cultures were treated with recombinant mCRP. Treatment of CECs with mCRP increased migration rate and monolayer permeability, while organ cultures treated with mCRP exhibited dramatically altered expression of inflammatory genes. Furthermore, in vitro complement activation assays suggest that complement activation on CECs can lead to the dissociation of pCRP into monomers on CECs. Our data indicate that 1) mCRP levels are elevated in individuals with the high-risk CFH genotype, 2) pro-inflammatory mCRP significantly affects endothelial cell phenotypes directly, both in vitro and ex vivo, and 3) MAC formation may be the driving force for accumulation of mCRP in the choriocapillaris. Altogether, this work suggests a role for mCRP in choroidal vascular dysfunction in AMD. Finally, we aimed to develop a reliable method for the production of human choroidal extracellular matrix (ECM) scaffolds to study CEC replacement strategies in an environment that closely resembles the native tissue. Human RPE/choroid tissue was treated sequentially with Triton X-100, SDS, and DNase to remove all native cells. While all cells were successfully removed from the tissue, collagen IV, elastin, and laminin remained, with preserved architecture of the acellular vascular tubes. The ECM scaffolds were then co-cultured with exogenous ECs to determine if the tissue can support cell growth and allow EC reintegration into the decellularized choroidal vasculature. Both monkey and human ECs took up residence in the choriocapillary tubes of the decellularized tissue. These data suggest that our decellularization methods are sufficient to remove all cellular material yet gentle enough to preserve tissue structure and allow for the optimization of cell replacement strategies. Together, these studies provide insight into the mechanism of AMD pathogenesis, suggest potential targets for drug therapies, and develop methods to study the replacement of CECs in more advanced cases of AMD.

Two of the most commonly diagnosed malignancies in men and women are cancers of the prostate and breast, respectively. Though many advances have been made in reducing the overall morbidity and mortality associated with these diseases, the high number of deaths that still occur emphasizes the need for safer and more effective therapeutic options. To this end, our lab was the first to describe the use of RNA aptamers to specifically deliver cytotoxic siRNAs to PSMA positive prostate cancer cells. This reagent, termed an aptamer-siRNA chimera, was shown to be an effective targeted cancer therapeutic upon intratumoral injection in a pre-clinical, xenograft, mouse model of prostate cancer. However, further work was needed to realize the full clinical potential of RNA aptamer-siRNA chimeras as a targeted therapeutic modality. The thesis laid out herein, describes work performed to optimize aptamer-siRNA technology in order to enable clinical translation and to increase the scope of this technology (i.e. increase the cancer types for which this technology can be used). We describe several improvements to our first generation PSMA aptamer-siRNA chimera which, include: decreasing the overall nucleotide content to aid in chemical synthesis, altering the siRNA structure to improve RNAi processing and addition of a 20kDa PEG moiety to increase pharmacokinetics/pharmacodynamics. All of these modifications lead to a more effective reagent at lower doses. Importantly, we demonstrate that our optimized reagent is now effective upon systemic administration in an in vivo mouse model of prostate cancer. In addition, we have also identified new aptamers to the receptor tyrosine kinase (RTK) EphA2. Given the broad expression of this RTK on various cancers, this work seeks to extend the scope of targeted aptamer therapeutics beyond that of prostate cancer. Finally, we demonstrate a novel aptamer selection methodology termed cell-internalization SELEX. This approach allowed us to select for aptamers that specifically targeted and internalize into HER2 expressing cells. This allowed us to readily translate all identified aptamers into aptamer-siRNA chimeras. We show that all chimeras tested were able to sensitize HER2+ breast cancer cells to low- dose cisplatin treatment. Taken together, the work described in this thesis significantly advances the field of targeted cancer therapeutics. Importantly, by demonstrating cancer cell-specific delivery of siRNA, our technology overcomes one of the most significant hurdles to the therapeutic use of siRNAs, delivery.